1. Paroxysmal Nocturnal
Hemoglobinuria
Debra Carnahan

2. Paroxysmal Nocturnal
Hemoglobinuria
First described by Dr. William Gull in 1866
Acquired chronic hemolytic disorder caused by complement-
mediated hemolysis of complement-sensitive erythrocytes
Affects approximately 1-10 individuals per 1,000,000
Mainly a disease of adults, although children and
adolescents also affected
Disorder affects both sexes almost equally
PNH is chronic, although spontaneous recoveries known to
occur
15% of patients underwent spontaneous remission of PNH at
10-20 years from diagnosis
Median survival time is 10-15 years from diagnosis

3. Etiology
Defect in PNH due to somatic mutations in pluripotent
hemopoietic stem cell of PIG-A gene located on X-
chromosome
PNH is an acquired hemolytic disorder.
But how is PNH acquired?
Apparently, the mechanism by which PNH is acquired is not
yet understood, so, the most fundamental cause of PNH
would be that which caused the gene mutation.
Some mutations are spontaneous and have no cause
Other mutations are induced by exposure to a mutagen such as
radiation or certain chemicals.

4. Pathophysiology
Mutations of PIG-A gene lead to block in
biosynthesis of glycosylphosphatidylinositol
(GPI) molecule
Some proteins attached to outer cell
membrane by GPI anchors
Physiological purpose of anchoring unknown –
other than anchoring itself
GPI-linked proteins easily released from cell
membrane
Proteins able to transfer between cells to some extent

5. Pathophysiology

6. Pathophysiology
Block in biosynthesis of GPI results in lack of GPI-
anchored proteins on surface of hemopoietic cells
in PNH patients
Deficiency of GPI-anchored CD59 (prolectin)
CD59 inhibits formation of membrane attack complex
(MAC) by binding to C8 and C9

7. Pathophysiology
Also deficiency of GPI-anchored CD55
(decay accelerating factor, DAF)
DAF accelerates decay of C3 convertases,
D4b2a and C3bBb, of classical and
alternative complement pathways,
respectively
About 30 GPI-anchored membrane
proteins recognized in human cells and,
of these, 20 have been shown to be
missing from blood cells of PNH patients

8. Pathophysiology

9. Pathophysiology
In PNH, these GPI-anchored regulatory proteins
either expressed in low numbers or totally absent
from RBCs, rendering them susceptible to
complement-mediated lysis
Types of mutation differ among patients
More than 100 PIG-A mutations reported
All result in total lack or severely diminished function of
PIG-A protein
Some mutations cause partial deficiency of GPI-
anchored proteins
Most mutations lead to non-functional
glycosyltransferase enzyme and complete absence of
GPI anchor synthesis

10. Pathophysiology
Defect occurs in all cell lines deriving
from mutated bone marrow cell:
leukocytes, platelets, as well as
RBCs, affected
Despite ability of GPI-anchored
proteins to transfer between cells, no
significant transfer seems to occur to
aberrant cells in PNH

11. Pathophysiology
Affected PNH cells of clonal origin, that is,
they appear to derive from one stem cell
In some patients, perhaps majority of patients,
2 or more defective clones arise
This could explain presence of cells with
variable expression levels of GPI-anchored
proteins
PNH I cells exhibit normal resistance to lysis
PNH II cells are 2-5 times more susceptible to
complement lysis
PNH III cells approximately 25 times more
susceptible to lysis

12. Pathophysiology
As aberrant cells more susceptible to
complement lysis, one might predict
complete removal of aberrant clone
In actuality, defective clone dominates over
normal cells
Reasons for this survival advantage not yet
known
Most important hypothesis is dual pathogenesis idea
in which “PNH clones expand relatively in association
with the elimination of GPI-positive hematopoietic
precursor cells”
Possibility that CD4+
lymphocytes with CD8+
cytotoxic-
T cells participate in negative selection of PNH clone

13. Pathophysiology
Aberrant clone may have limited
lifespan, which could account for its
spontaneous disappearance and
recovery of patient
These clones undergo senescence as a
result of telomere shortening
They succumb to autoimmune attack
Factors promoting their expansion
spontaneously remit

14. Pathophysiology
Bone marrow failure
Colony formation from erythroid progenitor
cells, and granulocyte-macrophage and
megakaryocyte precursor cells in peripheral
blood or bone marrow from PNH patients
decreased compared with that from healthy
individuals.
Decreased colony formation common to both
GPI-positive and -negative progentitor cells
from PNH patients due to proliferative defect,
not complement-mediated lysis of progenitor
cells

15. Pathophysiology
Bone marrow failure (cont’d)
Bone marrow failure syndromes include
aplasitic anemia (AA), PNH and
myelodysplastic syndrome (MDS)
Hypoplastic or aplastic bone marrow
– In PNH, marrow cellularity can vary from
hypocellular to hypercellular
Morphological abnormalities in cellular
marrow

16. Pathophysiology
Bone marrow failure (cont’d)
Bone marrow failure syndromes considered to
be pre-leukemic state
Up to 10% of patients with PNH develop acute
leukemia
Clinical and laboratory manifestations of PNH
disappear with onset of leukemia
Prognosis of acute leukemia arising from PNH very
poor
– Functional failure of bone marrow
– Abnormality in microenvironment as well as stem cell
abnormality

17. Clinical Presentation
PNH characterized by paroxysmal
intravascular hemolytic attacks
Brought on by:
Antecedent infections
Drug exposure
Trauma or other stress
Occurs spontaneously without identifiable
precipitating factor
Main manifestations:
Acute hemolysis may present as abdominal, lumbar
or sternal pain, or headache, fever, malaise
Thrombocytopenia can give rise to hemorrhagic
complications in some patients, but thromboses more
common

18. Clinical Presentation
Diminished hematopoiesis
Thrombotic tendency
Especially in abdominal veins
Hemolytic jaundice
As disease progresses:
Symptoms due to chronic uncompensated
hemolysis:
Weakness
Dyspnea
Pallor
Iron deficiency

19. Clinical Presentation
With time, severity relates to
proportion of complement-sensitive
cells and degree of marrow aplasia
Budd-Chiari syndrome (hepatic vein
thrombosis)
Intestinal infarction from repeated
hepatic and mesenteric vein thromboses
Infections from neutropenia and
leukocyte function defects
Exacerbates hemolysis

20. Clinical Presentation
With longstanding hemolysis, acute and
chronic renal failure may develop
Enlarged kidneys with excessive iron
deposits
Hematuria
Tubular malfunction
Diminished creatinine clearance
Hyposthenuria
Neurologic complications
Small venous occlusions

21. Clinical Presentation
Death usually result of:
Thromboembolism
Severe exacerbations of hemolysis
Infection or hemorrhage related to aplasia or
thrombocytopenia-associated hemorrhage

22. Lab Findings
Urinalysis:
Nocturnal hematuria/hemoglobinuria
Classic hallmark of PNH
Occurs in only about 25-50% of patients
Begins insidiously
Infrequent brown urine passed upon
awakening
Hemosiderinuria
Hemoglobin casts

23. Lab Findings

24. Lab Findings
Iron deficiency – due to hemolysis
Hemolytic anemia (hemoglobin 9-12 g/ml)
Diminished hemoglobin and platelets
Reticulocytosis with macrocytosis
Serum hemoglobin, unconjugated bilirubin
elevated
Haptoglobin low or absent
Granulocytes reduced
Bone marrow analysis:
Erythroid hyperplasia
Aplasia
28% of PNH patients presenting with AA

25. Lab Tests
Flow cytometry powerful tool for demonstrating whether cells
express or lack specific proteins on surface membranes
Gold standard for making diagnosis
Monoclonal antibodies to CD55 and/or CD59 utilized to
diagnose PNH and determine phenotype of PNH erythrocytes
With respect to sensitivity and specificity, flow cytometry
superior to old methods which employ complement-mediated
hemolysis in vitro
Ham’s test
Sugar-water test
Complement lysis sensitivity (CLS) test
Finding over 1% of CD59-negative cells considered positive
Blood transfusion and extensive hemolysis disturb results
http://www.unsolvedmysteries.oregonstate.edu/flow_cytometry_06.shtml

26. Lab Tests
Flow cytometry (cont’d)
Fluorescence-activated cell sorter
(FACS)
Type of flow cytometry
RBCs incubated with mouse monoclonal
antibodies to CD59 and after washing,
stained with fluorescein-conjugated
antimouse-IgG antibodies
Cells then analyzed using FACS

27. Lab Tests
Sucrose hemolysis test
Screening test
Serum pH lowered to about 6.2 and Mg2+ level adjusted
to 0.005 mol/L to achieve maximum sensitivity
Cells that are hemolyzed are the sensitive cells, and
those that remain intact are normal cells, indicating 2-3
subpopulations of RBCs in circulation
Test procedure:
One mL of patient citrated whole blood is added to 9.0 mL
of fresh sugar water reagent
Mix and incubate at room temperature for 30 minutes
If no hemolysis, then contradicts PNH diagnosis

28. Lab Tests
Ham or Acidified Serum Lysis test:
RBCs in PNH are lysed by complement when normal
serum is acidified or activated by alloantibodies
Procedure:
Use patient’s defibrinated whole blood
Set up test with patient’s RBCs and control RBCs in:
– Acidified patient’s serum
– Inactivated patient’s serum
– Patient’s serum
The test is positive if the patient’s RBCs:
– Hemolyze in their own serum
– Show increased hemolysis in acidified serum
– Do not hemolyze in the inactivated serum
The control cells demonstrate no hemolysis in all three
tubes

29. Lab Tests
Complement lysis sensitivity test
More precise
RBCs sensitized with potent lytic anti-i
antigen and hemolyzed with limiting
amounts of normal serum as source of
complement
This demonstrates 3 groups of RBCs in
PNH patients: PNH I cells, PNH II cells,
PNH III cells

30. Treatment
Treatment of PNH today still mainly
symptomatic
Blood transfusions used during periods
of severe hemolysis
Bone marrow transplantation only
available curative therapy
Risky
Matching transplants not easily available

31. Treatment
Medication
Anticoagulation therapy indicated during
venous thrombotic events
Immunosuppressive chemotherapy
– When pancytopenia present
– Stimulation of hematopoiesis in aplastic phase
High doses of corticosteroids considered
beneficial
Androgens stimulate erythropoiesis

32. Treatment
Medication (cont’d)
Complement inhibitor
– On March 16, 2007, FDA approved Soliris
(eculizumab) for treatment of PNH
– Results of studies showed treatment with
eculizumab produced dramatic reduction in
hemolysis, and days of hemoglobinuria each
month decreased

33. References
Noji, H., Tsutomu, S. (2002). A new aspect of the molecular
pathogenesis of paroxysmal nocturnal hemoglobinuria.
Hematology, 7 (4), 211-227.
Gordon-Smith, E. C., Marsh, J. C. W., & Tooze, J. A. (1999).
Clonal evolution of aplastic anaemia to myelodysplasia/acute
myeloid leukaemia and paroxysmal nocturnal
haemoglobinuria. Leukemia and Lymphoma, 33 (3-4), 231-
241.
Jarva, H., Meri, S. (1999). Paroxysmal nocturnal
haemoglobinuria: the disease and a hypothesis for a new
treatment. Scand J Immunol, 49, 119-125.
Araten, D. J., Swirsky, D., Karadimitris, A., Notaro, R.,
Khedoudja, N., Bessler, M., Thaler, H., Castro-Malaspina,
H., et al. (2001). Cytogenetic and morphological
abnormalities in paroxysmal nocturnal haemoglobinuria.
British Journal of Haematology, 115, 360-368.